Project Summary/Abstract A variety of gadolinium-based MRI contrast agents continue to be used clinically as non-specific extracellular agents but none report on specific metabolic indices of cancer. Although many responsive gadolinium agents have been reported in the literature, none are moving toward clinical approval largely because they are never truly silent and their response to biological events or tissue physiology is only modest. A novel class of MRI agents, the paramagnetic chemical exchange saturation transfer (paraCEST) agents, is better suited for the design of responsive MR agents because they are inherently more sensitive to changes in tissue physiology and metabolism. The extreme sensitivity of proton exchange rates to tissue environment makes these complexes an ideal platform for the design of responsive MR agents. Despite this potential, paraCEST agents have not shown much success in vivo at this point because 1) the field is small and only a few labs have tried to implement them in vivo and 2) their sensitivity in vivo is disappointingly low compare to in vitro. We are now convinced that the low in vivo sensitivity of paraCEST agents is because most agents were designed similar to Gd-based agents with an exchanging inner-sphere water molecule and this exchange adds substantial line- broadening to the tissue water signal by a T2exch mechanism. In this grant, we will focus on applications of paraCEST reporter molecules that lack an inner-sphere water exchange site thereby eliminating the detrimental T2exch effect on the bulk water signal. We will demonstrate the utility of these new agents for imaging common hallmarks of cancer in vivo before the end of the next 5 year grant cycle. In Aim 1, we will use paraCEST sensors to image the extracellular pH of multiple tumor types by detecting the frequency of the CEST signals arising from multiple ligand -OH groups. The agents we propose will have ?OH signals that shift frequency with changes in pH. In Aim 2, we will implement nitroimidazole-based MRI sensors for imaging hypoxic regions of tumors using agents that accumulate to high levels in hypoxic tissues. We have already shown that the Gd-derivatives of these agents are trapped only in hypoxic tumors and can be detected in T1- weighted images. The corresponding Yb- and Tm-derivatives will show a CEST signal that will also report the intracellular pH in those same hypoxic regions. In Aim 3, we outline a general strategy for improving the sensitivity of paraCEST agents by using pH-responsive block copolymer micelles as carriers that incorporate paraCEST centers in the hydrophobic block. The overarching goal of this grant is to implement responsive paraCEST agents in tumor-bearing animals at concentrations acceptable for translation to humans. We will show for the first time that two important hallmarks of cancer, pH and hypoxia, can be imaged routinely in vivo.